GSM5751373: Single mESC (fresh) S32_S32; Mus musculus; RNA-Seq
Cells were isolated with LCM in 6.4 μL of cell lysis buffer (1× GeneAmp PCR Buffer II [Thermo Fisher], 1.5 mM MgCl2 [Thermo Fisher], 0.5% sodium deoxycholate [SDc] [Nacalai Tesque], and 5 mM dithiothreitol [DTT] [Thermo Fisher] dissolved in deionized distilled water [Gibco] containing 4 U RNaseOUT [Thermo Fisher], 0.4 U porcine liver RNase inhibitor [Takara Bio], 0.05 mM dNTPs [Takara Bio], ERCC RNA Spike-In Mix 1 [Thermo Fisher], and 1.6 ng V1[dT]24 primer ) in the caps of PCR tubes, and spun down into the tubes by brief centrifugation.  Cells were then lysed at 70°C for 6 min, followed by the addition of 2.8 µL quenching buffer (25% Triton X-100 [Nacalai Tesque] and 1% bovine serum albumin [BSA] [Takara Bio]) and incubation at 70°C for 90 sec to quench the denaturing activity of SDc. The cDNA synthesis and amplification onward were performed as described previously with minor modifications [Kurimoto, K.et.al., Nucleic Acids Research, 34(5), e42.]. In brief, the first-strand cDNA was synthesized by SuperScript III [Thermo Fisher] and incubating at 50℃  for 5 min followed by heat inactivation at 70℃  for 10 min.  The excess RT primers were degraded by exonuclease I and incubating at 37℃ for 30 min followed by heat inactivation at 80℃ for 25 min. The first-strand cDNA product was poly dA-tailed by TdT and incubating at 37℃  for 1 min followed by heat inactivation at 70℃ for 10 min. Second-strand cDNA synthesis were performed by adding 9.5 μL reaction mixture of ExTaq, Hot Start version [Takara Bio] and V3[dT]24 primer in the ExTaq buffer with the following thermal cycling program (95℃  for 3 min, 50℃  for 2 min, and 72℃  for 3 min). cDNA amplification was performed by addingthe mixture of V1[dT]24 and ExTaq, Hot Start version [Takara Bio] in the ExTaq buffer with the following thermal cycling program (95℃  for 1 min; 20 cycles of 95℃  for 30 sec, 67℃  for 1 min and 72℃  for 3 min with a 6 sec elongation per cycle; and 72℃  for 10 min).  The amplified cDNA was purified with a 0.6× volume of AxyPrep MAG PCR clean-up reagent (Axygen) according to the manufacturer's instructions. Preparation of the libraries for SC3-seq was performed essentially as previously described (Nakamura et al., 2015)(Nakamura et al., 2017).  For Y-adapter sequencing, single-cell cDNAs were pre-amplified using the primers (V3[dT]24, V1[dT]24) and ExTaq HS with the thermal cycling program (95℃  for 3 min; 4 cycles of 95°C for 30 sec, 67℃  for 1 min and 72℃  for 5 min; 72℃  for 10 min). Pre-amplified cDNAs were then subjected to the primer-dimer removal, sonication, end polishing, and the size selection as in SC3-seq. Then, purified cDNA was dA-tailed in a 10 μL dA tailing mixture with incubation at 72℃  for 10 min. The dA-tailed product was subjected to the Y-shaped adaptor ligation by adding a mixture of Y-adaptor and NEB Next Quick Ligation Reaction Buffer and incubating at 20℃  for 15 min and at 72℃  for 20 min. The ligation product was purified with a 0.9×volume of AxyPrep reagent, and subjected to additional PCR amplification in 20 μL with the thermal cycling program (95℃  for 3 min, 55℃  for 1 min, and 72℃  for 1 min; 11 cycles of 95℃  for 30 sec, 60℃  for 1 min, and 72℃  for 1 min; and 72℃  for 3 min), followed by DNA purification with a 0.9×volume of AxyPrep reagent.